Photocleavable protecting groups and methods for their use

ABSTRACT

Novel compounds are provided which are useful as linking groups in chemical synthesis, preferably in the solid phase synthesis of oligonucleotides and polypeptides. These compounds are generally photolabile and comprise protecting groups which can be removed by photolysis to unmask a reactive group. The protecting group has the general formula Ar—C(R 1 )(R 2 )—O—C(O)— wherein:
         Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogous derivative thereof;   R 1  and R2 are independently H, optionally substituted alkyl, alkenyl or alkynyl, optionally substituted aryl or optionally substituted heteroaromatic, or a vinylogous derivative of the foregoing; and   X is a leaving group, a chemical fragment linked to Ar—C(R 1 )(R 2 )—O—C(O)— via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyl and R 1  and R 2  are H, X is not linked to Ar—C(R 1 )(R 2 )—O—C(O)— via a nitrogen atom. Preferred embodiments are those in which Ar is a fused polycyclic aromatic hydrocarbon and in which the substituents on Ar, R 1  and R 2  are electron donating groups. A particularly preferred protecting group is the “PYMOC” protecting group, pyrenylmethyloxycarbonyl, where Ar is pyrenyl and R 1  and R 2  are H.       

     Also provided is a method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, using the protected compounds described above.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 09/525,045, filed on Mar. 14, 2000, now U.S. Pat. No. 6,566,515;which is a continuation of U.S. patent application Ser. No. 08/812,005,filed on Mar. 5, 1997, now U.S. Pat. No. 6,147,205; which is acontinuation-in-part of U.S. patent application Ser. No. 08/630,148,filed Apr. 10, 1996, now U.S. Pat. No. 6,022,963; which is a regularapplication of Provisional Patent Application No. 60/008,684, filed Dec.15, 1995, all of which are incorporated herein by reference.

The present invention was made with U.S. Government support under ATPGrant No. 70NANB5H1031, and the government may have certain rights inthe invention.

BACKGROUND OF THE INVENTION

The present invention relates to the area of chemical synthesis. Moreparticularly, this invention relates to photolabile compounds, reagentsfor preparing the same and methods for their use as photocleavablelinkers and protecting groups, particularly in the synthesis of highdensity molecular arrays on solid supports.

The use of a photolabile molecule as a linker to couple molecules tosolid supports and to facilitate the subsequent cleavage reaction hasreceived considerable attention during the last two decades. Photolysisoffers a mild method of cleavage which complements traditional acidic orbasic cleavage techniques. See, e.g., Lloyd-Williams et al. (1993)Tetrahedron 49:11065-11133. The rapidly growing field of combinatorialorganic synthesis (see, e.g., Gallop et al. (1994) J. Med. Chem.37:1233-1251; and Gordon et al. (1994) J. Med. Chem. 37:1385-1401)involving libraries of peptides and small molecules has markedly renewedinterest in the use of photolabile linkers for the release of bothligands and tagging molecules.

A variety of ortho-benzyl compounds as photolabile protecting groupshave been used in the course of optimizing the photolithographicsynthesis of both peptides (see Fodor et al. (1994) Science 251:767-773)and oligonucleotides (see Pease et al. Proc. Natl. Acad. Sci. USA91:5022-5026). See PCT patent publication Nos. WO 90/15070, WO 92/10092,and WO 94/10128; see also U.S. patent application Ser. No. 07/971,181,filed 2 Nov. 1992, and Ser. No. 08/310,510, filed Sep. 22, 1994; Holmeset al. (1994) in Peptides: Chemistry, Structure and Biology (Proceedingsof the 13th American Peptide Symposium); Hodges et al. Eds.; ESCOM:Leiden; pp. 110-12, each of these references is incorporated herein byreference for all purposes. Examples of these compounds included the6-nitroveratryl derived protecting groups, which incorporate twoadditional alkoxy groups into the benzene ring. Introduction of anα-methyl onto the benzylic carbon facilitated the photolytic cleavagewith >350 nm UV light and resulted in the formation of a nitroso-ketone.

Photocleavable protecting groups and linkers should be stable to avariety of reagents (e.g., piperidine, TFA, and the like); be rapidlycleaved under mild conditions; and not generate highly reactivebyproducts. The present invention provides such protecting groups andmethods for their use in synthesizing high density molecular arrays.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, novel compounds areprovided which are useful for providing protecting groups in chemicalsynthesis, preferably in the solid phase synthesis of oligonucleotidesand polypeptides. These compounds are generally photolabile and compriseprotecting groups which can be removed by photolysis to unmask areactive group. The compounds have the general formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogous derivative thereof;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, optionally substituted aryl or optionally substitutedheteroaromatic, or a vinylogous derivative of the foregoing; and

X is a leaving group, a chemical fragment linked to Ar—C(R₁)(R₂)—O—C(O)—via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyland R₁═R₂═H, X is not linked to Ar—C(R₁)(R₂)—O—C(O)— via a nitrogenatom. Preferred embodiments are those in which Ar is a fused polycyclicaromatic hydrocarbon and in which the substituents on Ar, R₁ and R₂ areelectron donating groups. Particularly preferred protecting groups arethe “PYMOC” protecting group, pyrenylmethyloxycarbonyl, whereAr=1-pyrenyl and R₁═R₂═H, and the “ANMOC” protecting group,anthracenylmethyloxycarbonyl, where Ar=anthracenyl and R₁═R₂═H. Methodsare provided for preparing these compounds

This invention also provides reagents of the molecular formulaAr—C(R₁)(R₂)—O—C(O)—X, where Ar, R₁, and R₂ have the meanings ascribedabove, for incorporating the protecting group into the molecule desiredto be protected.

Another aspect of this invention provides a method of attaching amolecule with a reactive site to a support comprising the steps of:

(a) providing a support with a reactive site;

(b) binding a molecule to the reactive site, the molecule comprising amasked reactive site attached to a photolabile protecting group of theformula Ar—C(R₁)(R₂)—O—C(O)—, wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, or optionally substituted aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing;

to produce a derivatized support having immobilized thereon the moleculeattached to the photolabile protecting group; and

(c) removing the photolabile protecting group to provide a derivatizedsupport comprising the molecule with an unmasked reactive siteimmobilized thereon.

A related aspect of this invention provides a method of forming, fromcomponent molecules, a plurality of compounds on a support, eachcompound occupying a separate predefined region of the support, saidmethod comprising the steps of:

(a) activating a region of the support;

(b) binding a molecule to the region, said molecule comprising a maskedreactive site linked to a photolabile protecting group of the formulaAr—C(R₁)(R₂)—O—C(O)—, wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, or optionally substituted aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing;

(c) repeating steps (a) and (b) on other regions of the support wherebyeach of said other regions has bound thereto another molecule comprisinga masked reactive site linked to the photolabile protecting group,wherein said another molecule may be the same or different from thatused in step (b);

(d) removing the photolabile protecting group from one of the moleculesbound to one of the regions of the support to provide a region bearing amolecule with an unmasked reactive site;

(e) binding an additional molecule to the molecule with an unmaskedreactive site;

(f) repeating steps (d) and (e) on regions of the support until adesired plurality of compounds is formed from the component molecules,each compound occupying separate regions of the support.

The present invention also provides methods of performing chemicalreactions on a surface, by providing at least one chemical reactant onthe surface, and applying a coating to the surface. The coating providesan environment that is favorable to reaction of the chemical reactant.In a more preferred aspect, the invention provides a method ofactivating a functional group on a surface that is protected with aprotecting group of the invention. The method involves applying anucleophilic coating to the surface, and exposing the surface to lightto remove the protecting group.

The methods and compositions described herein find particular utility inthe synthesis of high density arrays of compounds, and particularlynucleic acids, on solid supports.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show syntheses of 5′-O-pyrenylmethyloxycarbonyl (“PYMOC”)protected deoxyribonucleoside 3′-O-cyanoethylphosphoramidites in asuitable form for coupling to a support.

FIG. 3 shows representative Ar groups that can be present in thecompounds of this invention.

FIG. 4 illustrates fluorescent scans of substrates subjected to dryfront-side exposure, coated front-side exposure and wet exposure(water/MeOH in a flow cell). Two coatings (0.1% Triton X-100/H₂O and0.2% Triton X-100/50% glycerol/H₂O) were tested and the results areshown in panels A and B, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

The term “alkyl” refers to a branched or straight chain acyclic,monovalent saturated hydrocarbon radical of one to twenty carbon atoms.

The term “alkenyl” refers to an unsaturated hydrocarbon radical whichcontains at least one carbon-carbon double bond and includes straightchain, branched chain and cyclic radicals.

The term “alkynyl” refers to an unsaturated hydrocarbon radical whichcontains at least one carbon-carbon triple bond and includes straightchain, branched chain and cyclic radicals.

The term “aryl” refers to an aromatic monovalent carbocyclic radicalhaving a single ring (e.g., phenyl) or two condensed rings (e.g.,naphthyl), which can optionally be mono-, di-, or tri-substituted,independently, with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio,lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl,hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl,lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl,trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, anddi-lower-alkylcarbamoyl. Alternatively, two adjacent positions of thearomatic ring may be substituted with a methylenedioxy or ethylenedioxygroup. Typically, electron-donating substituents are preferred.

The term “heteroaromatic” refers to an aromatic monovalent mono- orpoly-cyclic radical having at least one heteroatom within the ring,e.g., nitrogen, oxygen or sulfur, wherein the aromatic ring canoptionally be mono-, di- or tri-substituted, independently, with alkyl,lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl,thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy,mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl,lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl,lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano,tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl.For example, typical heteroaryl groups with one or more nitrogen atomsare tetrazoyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl), pyrrolyl(e.g., 2-pyrrolyl, 2-(N-alkyl)pyrrolyl), pyridazinyl, quinolyl (e.g.2-quinolyl, 3-quinolyl etc.), imidazolyl, isoquinolyl, pyrazolyl,pyrazinyl, pyrimidinyl, pyridonyl or pyridazinonyl; typical oxygenheteroaryl radicals with an oxygen atom are 2-furyl, 3-furyl orbenzofuranyl; typical sulfur heteroaryl radicals are thienyl, andbenzothienyl; typical mixed heteroatom heteroaryl radicals are furazanyland phenothiazinyl. Further the term also includes instances where aheteroatom within the ring has been oxidized, such as, for example, toform an N-oxide or sulfone.

The term “optionally substituted” refers to the presence or lack thereofof a substituent on the group being defined. When substitution ispresent the group may be mono-, di- or tri-substituted, independently,with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio,lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl,hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl,lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl,trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, anddi-lower-alkylcarbamoyl. Typically, electron-donating substituents suchas alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl,hydroxyl, thiol, amino, halo, lower-alkylthio, lower-alkoxy,mono-lower-alkylamino and di-lower-alkylamino are preferred.

The term “electron donating group” refers to a radical group that has alesser affinity for electrons than a hydrogen atom would if it occupiedthe same position in the molecule. For example, typical electrondonating groups are hydroxy, alkoxy (e.g. methoxy), amino, alkylaminoand dialkylamin0.

The term “leaving group” means a group capable of being displaced by anucleophile in a chemical reaction, for example halo, nitrophenoxy,pentafluorophenoxy, alkyl sulfonates (e.g., methanesulfonate), arylsulfonates, phosphates, sulfonic acid, sulfonic acid salts, and thelike.

“Activating group” refers to those groups which, when attached to aparticular functional group or reactive site, render that site morereactive toward covalent bond formation with a second functional groupor reactive site. For example, the group of activating groups which canbe used in the place of a hydroxyl group include —O(CO)Cl; —OCH₂Cl;—O(CO)OAr, where Ar is an aromatic group, preferably, a p-nitrophenylgroup; —O(CO)(ONHS); and the like. The group of activating groups whichare useful for a carboxylic acid include simple ester groups andanhydrides. The ester groups include alkyl, aryl and alkenyl esters andin particular such groups as 4-nitrophenyl, N-hydroxylsuccinimide andpentafluorophenol. Other activating groups are known to those of skillin the art.

“Chemical library” or “array” is an intentionally created collection ofdiffering molecules which can be prepared either synthetically orbiosynthetically and screened for biological activity in a variety ofdifferent formats (e.g., libraries of soluble molecules; and librariesof compounds tethered to resin beads, silica chips, or other solidsupports). The term is also intended to refer to an intentionallycreated collection of stereoisomers.

“Predefined region” refers to a localized area on a solid support whichis, was, or is intended to be used for formation of a selected moleculeand is otherwise referred to herein in the alternative as a “selected”region. The predefined region may have any convenient shape, e.g.,circular, rectangular, elliptical, wedge-shaped, etc. For the sake ofbrevity herein, “predefined regions” are sometimes referred to simply as“regions.” In some embodiments, a predefined region and, therefore, thearea upon which each distinct compound is synthesized smaller than about1 cm² or less than 1 mm². Within these regions, the molecule synthesizedtherein is preferably synthesized in a substantially pure form. Inadditional embodiments, a predefined region can be achieved byphysically separating the regions (i.e., beads, resins, gels, etc.) intowells, trays, etc.

“Solid support”, “support”, and “substrate” refer to a material or groupof materials having a rigid or semi-rigid surface or surfaces. In manyembodiments, at least one surface of the solid support will besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different compounds with, forexample, wells, raised regions, pins, etched trenches, or the like.According to other embodiments, the solid support(s) will take the formof beads, resins, gels, microspheres, or other geometric configurations.

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography,thick-layer (preparative) chromatography, distillation, or a combinationof these procedures. Specific illustrations of suitable separation andisolation procedures can be had by reference to the exampleshereinbelow. However, other equivalent separation or isolationprocedures can, of course, also be used.

A “channel block” is a material having a plurality of grooves orrecessed regions on a surface thereof. The grooves or recessed regionsmay take on a variety of geometric configurations, including but notlimited to stripes, circles, serpentine paths, or the like. Channelblocks may be prepared in a variety of manners, including etchingsilicon blocks, molding or pressing polymers, etc.

This invention provides novel compounds which are useful for providingprotecting groups in chemical synthesis, preferably in the solid phasesynthesis of oligonucleotides and polypeptides and high density arraysthereof. These compounds are generally photolabile and compriseprotecting groups which can be removed by photolysis to unmask areactive group. The compounds have the general formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogous derivative thereof,

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, optionally substituted aryl or optionally substitutedheteroaromatic, or a vinylogous derivative of the foregoing; and

X is a leaving group, a chemical fragment linked to Ar—C(R₁)(R₂)—O—C(O)—via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyland R₁═R₂═H, X is not linked to Ar—C(R₁)(R₂)—O—C(O)— via a nitrogenatom.

Preferred embodiments are those in which Ar is a fused polycyclicaromatic hydrocarbon and in which the substituents on Ar, R₁ and R₂ areelectron donating groups, such as alkoxy groups, particularly one ormore methoxy groups. Examples of electron donating carrying R₁ and R₂groups are methyl, substituted phenyl groups, e.g., o- orp-methoxyphenyl; 2,6-dimethoxyphenyl; 2,3-dimethoxyphenyl;3,5-dimethoxyphenyl and the like. Other R₁ and R₂ groups include9-anthracenyl or 1-pyrenyl. Particularly preferred protecting groups arethe “PYMOC” protecting group, 1-pyrenylmethyloxycarbonyl, whereAr=1-pyrenyl and R₁═R₂═H, and the “ANMOC” protecting group,anthracenylmethyloxycarbonyl, where Ar=anthracenyl (e.g. 9-anthracenyl)and R₁═R₂═H.

Representative fused polycyclic aromatic hydrocarbons includenaphthalene, phenanthrene, anthracene, benzoanthracene,dibenzoanthracene, heptalene, acenaphthalene, acephenanthrene,triphenylene, pyrene, fluorene, phenalene, naphthacene, picene,perylene, pentaphenylene, pyranthrene, fullerenes (including C₆₀ andC₇₀), and the like. A representative vinylogously substituted derivativeof an aromatic hydrocarbon is styrene.

The invention also provides reagents of the molecular formulaAr—C(R₁)(R₂)—O—C(O)—X, where Ar, R₁, and R₂ have the meanings ascribedabove, for incorporating the protecting group into the molecule desiredto be protected. X can be any suitable leaving group such as halo,oxycarbonyl, imidazolyl, pentafluorophenoxy and the like, which iscapable of reacting with a nucleophilic group such as hydroxy, amino,alkylamino, thio and the like on the molecule being protected. Thus, thereagents comprising the protecting groups Ar—C(R₁)(R₂)—O—C(O)— disclosedherein can be used in numerous applications where protection of areactive nucleophilic group is required. Such applications include, butare not limited to polypeptide synthesis, both solid phase and solutionphase, oligo- and polysaccharide synthesis, polynucleotide synthesis,protection of nucleophilic groups in organic syntheses of potentialdrugs, etc.

The invention also provides compositions of the molecular formulaAr—C(R₁)(R₂)—O—C(O)—M, where Ar, R₁ and R₂ have the meaning outlinedabove and M is any other chemical fragment. Preferably, M will be amonomeric building block that can be used to make a macromolecule. Suchbuilding blocks include amino acids, peptides, polypeptides, nucleicacids, nucleotides, nucleosides, monosaccharides, and the like.Preferred nucleosides are ribonucleosides and deoxyribonucleosides suchas adenosine, deoxyadenosine, cytidine, deoxycytidine, thymidine,uracil, guanosine and deoxyguanosine as well as oligonucleotidesincorporating such nucleosides. Preferably, the building block is linkedto the photolabile protecting group via a hydroxy or amine group. Whennucleotide and oligonucleotide compositions are used, with theprotecting groups of this invention, the protecting groups arepreferably incorporated into the 3′-OH or the 5′-OH of the nucleoside.Other preferred compounds are protected peptides, proteins,oligonucleotides and oligodeoxyribonucleotides. Small organic molecules,proteins, hormones, antibodies and other such species havingnucleophilic reactive groups can be protected using the protectinggroups disclosed herein.

The use of nucleoside and nucleotide analogs is also contemplated bythis invention to provide oligonucleotide or oligonucleoside analogsbearing the protecting groups disclosed herein. Thus the termsnucleoside, nucleotide, deoxynucleoside and deoxynucleotide generallyinclude analogs such as those described herein. These analogs are thosemolecules having some structural features in common with a naturallyoccurring nucleoside or nucleotide such that when incorporated into anoligonucleotide or oligonucleoside sequence, they allow hybridizationwith a naturally occurring oligonucleotide sequence in solution.Typically, these analogs are derived from naturally occurringnucleosides and nucleotides by replacing and/or modifying the base, theribose or the phosphodiester moiety. The changes can be tailor made tostabilize or destabilize hybrid formation or enhance the specificity ofhybridization with a complementary nucleic acid sequence as desired.

Analogs also include protected and/or modified monomers as areconventionally used in oligonucleotide synthesis. As one of skill in theart is well aware oligonucleotide synthesis uses a variety ofbase-protected deoxynucleoside derivatives in which one or more of thenitrogens of the purine and pyrimidine moiety are protected by groupssuch as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.Specific monomeric building blocks which are encompassed by thisinvention include base protected deoxynucleoside H-phosphonates anddeoxynucleoside phosphoramidites.

For instance, structural groups are optionally added to the ribose orbase of a nucleoside for incorporation into an oligonucleotide, such asa methyl, propyl or allyl group at the 2′-O position on the ribose, or afluoro group which substitutes for the 2′-O group, or a bromo group onthe ribonucleoside base. 2′-O-methyloligoribonucleotides (2′-O-MeORNs)have a higher affinity for complementary nucleic acids (especially RNA)than their unmodified counterparts. 2′-O-MeORNA phosphoramidite monomersare available commercially, e.g., from Chem Genes Corp. or GlenResearch, Inc. Alternatively, deazapurines and deazapyrimidines in whichone or more N atoms of the purine or pyrimidine heterocyclic ring arereplaced by C atoms can also be used.

The phosphodiester linkage, or “sugar-phosphate backbone” of theoligonucleotide analogue can also be substituted or modified, forinstance with methyl phosphonates or O-methyl phosphates. Anotherexample of an oligonucleotide analogue for purposes of this disclosureincludes “peptide nucleic acids” in which a polyamide backbone isattached to oligonucleotide bases, or modified oligonucleotide bases.Peptide nucleic acids which comprise a polyamide backbone and the basesfound in naturally occurring nucleosides are commercially availablefrom, e.g., Biosearch, Inc. (Bedford, Mass.).

Nucleotides with modified bases can also be used in this invention. Someexamples of base modifications include 2-aminoadenine, 5-methylcytosine,5-(propyn-1-yl)cytosine, 5-(propyn-1-yl)uracil, 5-bromouracil, and5-bromocytosine which can be incorporated into oligonucleotides in orderto increase binding affinity for complementary nucleic acids. Groups canalso be linked to various positions on the nucleoside sugar ring or onthe purine or pyrimidine rings which may stabilize the duplex byelectrostatic interactions with the negatively charged phosphatebackbone, or through hydrogen bonding interactions in the major andminor groves. For example, adenosine and guanosine nucleotides can besubstituted at the N² position with an imidazolyl propyl group,increasing duplex stability. Universal base analogues such as3-nitropyrrole and 5-nitroindole can also be included. A variety ofmodified oligonucleotides and oligonucleotide analogs suitable for usein this invention are described in, e.g., “Antisense Research andApplications”, S. T. Crooke and B. LeBleu (eds.) (CRC Press, 1993) and“Carbohydrate Modifications in Antisense Research” in ACS Symp. Ser.#580, Y. S. Sanghvi and P. D. Cook (eds.) ACS, Washington, D.C. 1994).

Compounds of this invention can be prepared by carbonylating an aromaticcarbinol of the general formula Ar—C(R₁)(R₂)—OH with a carbonylationreagent such as for example, phosgene (COCl₂), carbonyldiimidazole orpentafluorophenoxy chloroformate and the like to provideAr—C(R₁)(R₂)—O—C(O)—X where X is a leaving group derived from thecarbonylating reagent (Cl, if phosgene was used, pentafluorophenoxy, ifpentafluorophenoxy chloroformate was used, etc.). This intermediate,Ar—C(R₁)(R₂)—O—C(O)—X is then reacted with a molecule M carrying anucleophilic group whose protection is desired to yield a protectedbuilding block Ar—C(R₁)(R₂)—O—C(O)-M. Representative aromatic carbinolsare pyrenemethanol, naphthalenemethanol, anthracenemethanol,perylenemethanol and the like. Such aromatic carbinols are availablefrom commercial suppliers such as Aldrich Chemical Co., Milwuakee, Wis.Alternatively, they may also be obtained from precursor aromatichydrocarbons by acylation under Friedel-Crafts conditions with acidchlorides and anhydrides and subsequent reduction of the carbonyl groupthus added to a carbinol.

Alternatively, one may first carbonylate the group on the molecule beingprotected with a carbonylation reagent, such as one described above, andsubsequently displace the leaving group X thus inserted with thehydroxyl group of the aromatic carbinol. In either procedure, onefrequently uses a base such as triethylamine or diisopropylethylamineand the like to facilitate the displacement of the leaving group.

One of skill in the art will recognize that the protecting groupsdisclosed herein can also be attached to species not traditionallyconsidered as “molecules”. Therefore, compositions such as solidsurfaces (e.g., paper, nitrocellulose, glass, polystyrene, silicon,modified silicon, GaAs, silica and the like), gels (e.g., agarose,sepharose, polyacrylamide and the like) to which the protecting groupsdisclosed herein are attached are also contemplated by this invention.

The protecting groups of this invention are typically removed byphotolysis, i.e., by irradiation, though in selected cases it may beadvantageous to use acid or base catalyzed cleavage conditions.Generally irradiation is at wavelengths greater than about 340 nm,preferably at about 365 nm. The photolysis is usually conducted in thepresence of hydroxylic or protic solvents, such as aqueous, alcoholic ormixed aqueous-alcoholic or mixed aqueous-organic solvent mixtures.Alcoholic solvents frequently used include methanol and ethanol. Thephotolysis medium may also include nucleophilic scavengers such ashydrogen peroxide. Photolysis is frequently conducted at neutral orbasic pH.

This invention also provides a method of attaching a molecule with areactive site to a support, comprising the steps of:

(a) providing a support with a reactive site;

(b) binding a molecule to the reactive site, said first moleculecomprising a masked reactive site attached to a photolabile protectinggroup of the formula Ar—C(R₁)(R₂)—O—C(O)—, wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, or optionally substituted aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing;

to produce a derivatized support having immobilized thereon the moleculeattached to the photolabile protecting group; and

(c) removing the photolabile protecting group to provide a derivatizedsupport comprising the molecule with an unmasked reactive siteimmobilized thereon.

As one of skill will recognize, the process can be repeated to generatea compound comprising a chain of component molecules attached to thesolid support. In a “mix and match” approach, the photolabile protectinggroups may be varied at different steps in the process depending on theease of synthesis of the protected precursor molecule. Alternatively,photolabile protecting groups can be used in some steps of the synthesisand chemically labile (e.g. acid or base sensitive groups) can be usedin other steps, depending for example on the availability of thecomponent monomers, the sensitivity of the substrate and the like. Thismethod can also be generalized to be used in preparing arrays ofcompounds, each compound being attached to a different and identifiablesite on the support as is disclosed in U.S. Pat. Nos. 5,143,854,5,384,261, 5,424,186 5,445,934 and copending U.S. patent applicationSer. No. 08/376,963, filed Jan. 23, 1995, incorporated herein byreference for all purposes.

Thus, a related aspect of this invention provides a method of forming,from component molecules, a plurality of compounds on a support, eachcompound occupying a separate predefined region of the support, saidmethod comprising the steps of:

(a) activating a region of the support;

(b) binding a molecule to the region, said molecule comprising a maskedreactive site linked to a photolabile protecting group of the formulaAr—C(R₁)(R₂)—O—C(O)—, wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, or optionally substituted aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing;

(c) repeating steps (a) and (b) on other regions of the support wherebyeach of said other regions has bound thereto another molecule comprisinga masked reactive site linked to the photolabile protecting group,wherein said another molecule may be the same or different from thatused in step (b);

(d) removing the photolabile protecting group from one of the moleculesbound to one of the regions of the support to provide a region bearing amolecule with an unmasked reactive site;

(e) binding an additional molecule to the molecule with an unmaskedreactive site;

(f) repeating steps (d) and (e) on regions of the support until adesired plurality of compounds is formed from the component molecules,each compound occupying separate regions of the support.

A related method of forming a plurality of compounds on predefinedregions of a support involves binding a molecule with a reactive siteprotected with a chemically labile protecting group to an activatedregion of the support and chemically removing the chemically labileprotecting group to reveal the reactive site. The reactive site is thenprotected with a photolabile protecting group of this invention. Thisprocess is repeated for other regions of the support with othermolecules as desired to provide a support having molecules with reactivesites protected by photolabile protecting groups on separate regions ofthe support. Reactive sites can be unmasked by removing the photolabilegroup from selected regions and coupled to additional molecules withphotolabile protecting groups as described earlier to build up arrays ofcompounds on the support. Again, in a “mix and match” approach, monomerswith chemically labile protecting groups can be attached to a reactivesite on the substrate (i.e., on the support itself when the first layerof monomers is being assembled or subsequently onto an already attachedmonomer whose reactive site has been unmasked) and these chemicallylabile protecting groups can be replaced by a photolabile protectinggroups of this invention. The replacement is accomplished by removingthe chemically labile protecting group under conditions which do notaffect any photolabile groups which may be on the support. This thenreveals an unmasked reactive site on the monomer which had carried thechemically labile protecting group and this unmasked reactive site isreacted with a reagent of the formula Ar—C(R₁)(R₂)—O—C(O)—X, where X isa leaving group. Thereby, this region of the support is protected by aphotolabile protecting group which can be selectively removed by lightdirected systems described in U.S. Pat. Nos. 5,143,854, 5,384,261,5,424,186 and 5,445,934 and further described below. This method isparticularly useful when the monomers are more readily availablecarrying chemically labile protecting groups than the photolabileprotecting groups described herein. It will be recognized that anymethod of forming a chain of compounds or an array of compounds on asupport using in at least one step a protecting group/reagent orcompound of this invention is within the scope of the methods thisinvention.

Generally, these methods involve sequential addition of monomers tobuild up an array of polymeric species on a support by activatingpredefined regions of a substrate or solid support and then contactingthe substrate with a protected monomer of this invention (e.g., a PYMOCprotected nucleoside or amino acid). It will be recognized that theindividual monomers can be varied from step to step. A common support isa glass or silica substrate as is used in semiconductor devices.

The predefined regions can be activated with a light source, typicallyshown through a screen such as a photolithographic mask similar to thetechniques used in integrated circuit fabrication. Other regions of thesupport remain inactive because they are blocked by the mask fromillumination and remain chemically protected. Thus, a light patterndefines which regions of the support react with a given monomer. Theprotected monomer reacts with the activated regions and is immobilizedtherein. The protecting group is removed by photolysis and washed offwith unreacted monomer. By repeatedly activating different sets ofpredefined regions and contacting different monomer solutions with thesubstrate, a diverse array of polymers of known composition at definedregions of the substrate can be prepared. Arrays of 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹² or more different polymers can be assembled on thesubstrate. The regions may be 1 mm² or larger, typically 10 μm² and maybe as small as 1 μm². These regions are also referred to herein as“features.”

In the preferred methods of preparing these arrays, contrast betweenfeatures may be enhanced through the front side exposure of thesubstrate. By “front side exposure” is meant that the activation lightis incident upon the synthesis side of the substrate, contacting thesynthesis side of the substrate prior to passing through the substrate.Front side exposure reduces effects of diffraction or divergence byallowing the mask to be placed closer to the synthesis surface.Additionally, and perhaps more importantly, refractive effects from thelight passing through the substrate surface, prior to exposure of thesynthesis surface, are also reduced or eliminated by front-sideexposure. Front side exposure is described in substantial detail in U.S.patent application Ser. No. 08/634,053 filed Apr. 17, 1996, incorpratedherein by reference.

As noted previously, however, the efficiency of photolysis of thepreferred photolabile protecting groups of the present invention isimproved when such photolysis is carried out in the presence ofnucleophilic solvents, such as water or methanol. This presents a uniqueproblem where front side photolysis is used. Specifically, as the frontside of the substrate is exposed to the activation radiation, a flowcell cannot be used to maintain the desired nucleophilic environmentduring such photolysis. Accordingly, in preferred aspects,light-directed synthesis methods employing the protecting groups of thepresent invention is carried out by providing a thin aqueous film orcoating on the synthesis surface of the substrate. The presence of thisthin film or coating allows one to control the local environment on thesynthesis surface, i.e., to provide conditions that are favorable forthat synthesis. By “conditions favorable to reaction” is meantconditions that result in an improvement of reaction efficiency of agiven chemical reactant or reactants, over reactions not performed inthat environment, e.g., reaction rate, yield, or both. For example, forsynthesis methods employing the protecting groups described herein,coatings may be applied that provide a nucleophic environment which isfavorable to photolysis of the protecting group, and which therebypromotes efficient synthesis. The use of such coatings also permits thefront side exposure of the substrate surface. This method may also beperformed in reacting more than one chemical reactant, by applying bothreactants on the surface prior to coating, or by adding the secondreactant after the coating or as an element of the coating.

Generally, a thin film or coating of aqueous solution can be applied tothe synthesis surface of a substrate that is bearing the protectinggroups of the invention, e.g., that has been subjected to previoussynthesis steps. Application of the coating may be carried out bymethods that are well known in the art. For example, spin-coatingmethods may be utilized where the substrate is spun during applicationof the coating material to generate a uniform coating across the surfaceof the substrate. Alternative application methods may also be used,including simple immersion, spray coating methods and the like.

Aqueous solutions for use as coating materials typically include, e.g.,low molecular weight poly-alcohols, such as ethylene glycol, propyleneglycol, glycerol and the like. These solutions are generally hygrophilicand provide nucleophilic hydroxyl groups which will also support thephotolysis reaction. The poly-alcohols also increase the viscosity ofthe solution, which can be used to control the thickness of the coating.Higher molecular weight poly-alcohols, i.e., polyvinyl alcohol, may alsobe used to adjust the viscosity of the coating material.

Generally, preferred substrates have relatively hydrophobic surfaces. Assuch, the aqueous coating solution may also include an appropriatesurfactant, e.g., from about 0.01 to about 10% v/v to permit spreadingand adhesion of the film upon the substrate surface. Such surfactantsgenerally include those that are well known in the art, including, e.g.,Triton X-100, Tween-80, and the like. In addition to promoting thespreading and adhesion of the coating to the substrate, addition of athese non-volatile solutes within the coating solution can limit theamount of evaporation of the film and promote its longevity.

The methods described herein may also employ component moleculescomprising a masked reactive site attached to a photolabile protectinggroup of the formula

Ar—C(R₁)(R₂)—, wherein Ar, R₁, and R₂ have the meanings ascribedearlier. In such cases, the protecting group is attached to a reactivesite that is not an amine and is removed by photolysis.

The solid substrate or solid support may be of any shape, although theypreferably will be roughly spherical. The supports need not necessarilybe homogenous in size, shape or composition, although the supportsusually and preferably will be uniform. In some embodiments, supportsthat are very uniform in size may be particularly preferred. In anotherembodiment, two or more distinctly different populations of solidsupports may be used for certain purposes.

Solid supports may consist of many materials, limited primarily bycapacity for derivatization to attach any of a number of chemicallyreactive groups and compatibility with the synthetic chemistry used toproduce the array and, in some embodiments, the methods used for tagattachment and/or synthesis. Suitable support materials typically willbe the type of material commonly used in peptide and polymer synthesisand include glass, latex, polyethylene glycol, heavily cross-linkedpolystyrene or similar polymers, gold or other colloidal metalparticles, and other materials known to those skilled in the art. Thechemically reactive groups with which such solid supports may bederivatized are those commonly used for solid phase synthesis of thepolymer and thus will be well known to those skilled in the art, i.e.,carboxyls, amines, and hydroxyls.

To improve washing efficiencies, one can employ nonporous supports orother solid supports less porous than typical peptide synthesissupports; however, for certain applications of the invention, quiteporous beads, resins, or other supports work well and are oftenpreferable. One such support is a resin in the form of beads. Ingeneral, the bead size is in the range of 1 nm to 100 μm, but a moremassive solid support of up to 1 mm in size may sometimes be used.Particularly preferred resins include Sasrin resin (a polystyrene resinavailable from Bachem Bioscience, Switzerland); and TentaGel S AC,TentaGel PHB, or TentaGel S NH₂ resin (polystyrene-polyethylene glycolcopolymer resins available from Rappe Polymere, Tubingen, Germany).Other preferred supports are commercially available from and describedby Novabiochem, La Jolla, Calif.

In other embodiments, the solid substrate is flat, or alternatively, maytake on alternative surface configurations. For example, the solidsubstrate may contain raised or depressed regions on which synthesistakes place. In some embodiments, the solid substrate will be chosen toprovide appropriate light-absorbing characteristics. For example, thesubstrate may be a polymerized Langmuir Blodgett film, functionalizedglass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of avariety of gels or polymers such as (poly)tetrafluorethylene,(poly)vinylidendifluoride, polystyrene, polycarbonate, or combinationsthereof. Other suitable solid substrate material will be readilyapparent to those of skill in the art. Preferably, the surface of thesolid substrate will contain reactive groups, which could be carboxyl,amino, hydroxyl, thiol, or the like. More preferably, the surface willbe optically transparent and will have surface Si—OH functionalities,such as are found on silica surfaces.

The photolabile protecting groups and protected monomers disclosedherein can also be used in bead based methods of immobilization ofarrays of molecules on solid supports.

A general approach for bead based synthesis is described in copendingapplication Ser. No. 07/762,522 (filed Sep. 18, 1991); Ser. No.07/946,239 (filed Sep. 16, 1992); Ser. No. 08/146,886 (filed Nov. 2,1993); Ser. No. 07/876,792 (filed Apr. 29, 1992) and PCT/US93/04145(filed Apr. 28, 1993), Lam et al. (1991) Nature 354:82-84; PCTapplication no. 92/00091 and Houghten et al, (1991) Nature 354:84-86,each of which is incorporated herein by reference for all purposes.

Other methods of immobilization of arrays of molecules in which thephotocleavable protecting groups of this invention can be used includepin based arrays and flow channel and spotting methods.

Photocleavable arrays also can be prepared using the pin approachdeveloped by Geysen et al. for combinatorial solid-phase peptidesynthesis. A description of this method is offered by Geysen et al., J.Immunol. Meth. (1987) 102:259-274, incorporated herein by reference.

Additional methods applicable to library synthesis on a single substrateare described in co-pending application Ser. No. 07/980,523, filed Nov.20, 1992, and Ser. No. 07/796,243, filed Nov. 22, 1991, incorporatedherein by reference for all purposes. In the methods disclosed in theseapplications, reagents are delivered to the substrate by either (1)flowing within a channel defined on predefined regions or (2) “spotting”on predefined regions. However, other approaches, as well ascombinations of spotting and flowing, may be employed. In each instance,certain activated regions of the substrate are mechanically separatedfrom other regions when the monomer solutions are delivered to thevarious reaction sites. Photocleavable linkers are particularly suitablefor this technology as this delivery method may otherwise result in poorsynthesis fidelity due to spreading, reagent dilution, inaccuratedelivery, and the like. By using a photocleavable linker, rather than aconventional acid-cleavable linker, the purest material can beselectively cleaved from the surface for subsequent assaying or otherprocedures. More specifically, masks can be used when cleaving thelinker to ensure that only linker in the center of the delivery area(i.e., the area where reagent delivery is most consistent andreproducible) is cleaved. Accordingly, the material thus selectivelycleaved will be of higher purity than if the material were taken fromthe entire surface.

Typically, the molecules used in this method will be the monomericcomponents of complex macromolecules. These monomeric components can besmall ligand molecules, amino acids, nucleic acids, nucleotides,nucleosides, monosaccharides and the like, thereby allowing one tosynthesize arrays of complex macromolecules or polymeric sequences, suchas polypeptides, nucleic acids and synthetic receptors, on the solidsupport.

This invention discloses new nucleoside phosphoramidite monomers with1-pyrenylmethyloxy-carbonyl (“PYMOC”) 5′-protecting groups. They arephotolytically cleaved under irradiation at wavelengths greater thanabout 340 nm, preferably at about 365 nm, in the presence of methanol,water, or water-solvent mixtures and/or with nucleophilic scavengerssuch as hydrogen peroxide at neutral or basic pH. The rate of photolysisis similar to that observed for the MeNPOC group. However, the yield ofPYMOC photo-removal is much higher (˜95%), so that the use of thesemonomers for photochemical synthesis of oligonucleotides leads to higherstepwise cycle yields and therefore higher-purity oligomers.

The 1-pyrenylmethyloxycarbonyl group described here can be used for theprotection of alcohols. The photolysis of PYMOC is faster than that ofthe 1-pyrenylmethyl group, so it would also be a superiorphoto-removable protecting group for phosphates, carboxylates, amines,thiols, etc.

Other “benzylic” oxycarbonyls may have similar or better efficiency thanthe PYMOC group. A general formula would be:

where Ar is an optionally substituted fused polycyclic aryl or anoptionally substituted heteroaromatic group or a vinylogouslysubstituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, optionally substituted aryl, optionally substitutedheteroaromatic, or vinylogously substituted derivatives of theforegoing. Preferred embodiments are those in which Ar is a fusedpolycyclic aromatic hydrocarbon. FIG. 2 shows representative examples.Preferred substituents on the aromatic hydrocarbons would beelectron-donating groups that stabilize an incipient excited statebenzyl carbocation.

Other embodiments of the PYMOC photogroup, for example, include at leastone additional substituent at the a position, such as a methyl group ora methoxy-substituted phenyl. These substituents will increasephotosolvolysis efficiency, and improve the selectivity for the5′-hydroxyl in the preparation of the monomer 5′-protected nucleoside.

EXAMPLES Synthesis of5′-O-PYMOC-2′-DEOXYNUCLEOSIDE-3′-O-(N,N-DIISOPROPYL)CYANOETHYLPHOSPHORAMIDITES

All chemical reagents used were procured from commercial sources(Aldrich Chemical Co., Milwaukee, Wis. and Sigma Chemical Co.,Milawaukee, Wis.). Intermediates and products were identified by massspectrometry, ¹H-NMR, and ³¹P-NMR.

Abbreviations:

DIEA—Diethyl isopropylamine

NHS—N-hydroxysuccinimide

THF—Tetrahydrofuran

MeNPOC—methylnitropiperonyloxycarbonyl

TEA—Triethylamine

DMAP—4-Dimethylaminopyridine

Pentafluorophenyl Chloroformate

Pentafluorophenol (30 g; 163 mmol) and triethylamine (20 g, 200 mmol)were combined in 200 ml dry THF, and then added dropwise to a stirringsolution of phosgene (20 g; 200 mmol) in 100 ml of toluene at 0° C.After 2 hours, the solution was filtered and evaporated to give thecrude product as an oil, which was recrystallized from hexane to obtain30 g (75%) pure pentafluorophenyl chloroformate.

5′-O-(1-Pyrenylmethyl)- and 5′-O-(9-anthracenylmethyl)oxycarbonyl-2′-deoxynucleosides

The following general procedure was used to prepare 5′-PYMOC-derivativesof thymidine, N-4-isobutyryl-2′-deoxycytidine,N-7-isobutyryl-2′-deoxyadenosine, and N-4-isobutyryl-2′-deoxyguanosine;and 5′-ANMOC isobutyryl-2′-deoxyadenosine:

The base-protected nucleoside (20 mmol) was dried by co-evaporating 3times with 50 ml dry pyridine, then dissolved in 20 ml CH₂Cl₂ and 10 mldimethylsulfoxide (DMSO) containing 1.7 ml (21 mmol) pyridine. Theresulting solution was cooled to −10° C. under argon, and 5 g (20 mmol)of pentafluorophenyl chloroformate was added all at once with stirring.After an additional 2-3 hours stirring at −110° C., the reaction mixturewas analyzed by TLC or HPLC to determine the extent of conversion.Additional quantities of pentafluorophenyl chloroformate and pyridine(˜0.4-1.0 mmol each) were then added, as needed, until the nucleosidewas completely converted to the 5′-O-pentafluorophenoxycarbonylderivative. Although isolable, at this point the intermediate wasusually converted directly to the PYMOC or ANMOC-derivative, in situ, bythe addition of 1-pyrenemethanol (6 g, 26 mmol) or 9-anthracenemethanol,followed by 10 ml of triethylamine and 0.25 g (2 mmol) ofN,N-dimethyl-aminopyridine, and stirring overnight at room temperature.About 50-100 ml of CH₂Cl₂ was then added, and in the case of thymidine,the pure PYMOC-derivative precipitated and could be collected byfiltration. Otherwise, the solution was washed twice with 5% aqueousNaHCO₃, once with saturated NaCl, dried with Na₂SO₄, and evaporated todryness. The crude material was finally purified by flash chromatography(silica gel, 2:8 ethyl acetate-CH₂Cl₂/1-6% methanol gradient) to obtainthe pure 5′-PYMOC or ANMOC nucleoside products in ˜75% yield. The purityof the nucleosides was determined by HPLC, ¹H-NMR, mass spectrometry andelemental analysis (CHN).

5′-O-(1-Pyrenylmethyl)oxycarbonyl-2′-deoxynucleoside-3′-O—(N,N-diisopropyl)Cyanoethylphosphoramidites

On a 12 mmol scale, the 5′-PYMOC and ANMOC nucleosides were first driedby co-evaporation with dry pyridine, and then dissolved or suspended in50 ml of dry CH₂Cl₂. Then2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphorodiamidite (4.4 g; 14.5mmol) and N,N-diisopropylammonium tetrazolide (1 g; 6 mmol) were added,and the mixture was left stirring under argon overnight. The solutionwas washed twice with 10% aqueous NaHCO₃, once with saturated NaCl,dried with Na₂SO₄, and then evaporated to dryness. The crude productswere purified by flash chromatography (silica gel, eluting with a 1-5%methanol gradient in 2:8 ethyl acetate-CH₂Cl₂ containing 0.5%triethylamine) to obtain the pure phosphoramidites in ˜80% yield. Puritywas established by HPLC, ¹H, ³¹P-NMR, mass spectrometry and elementalanalysis (CHN)

Table 1 compares the efficiency of photolytic cleavage of PYMOC andANMOC protected nucleosides to MeNPOC (methylnitropiperonyloxycarbonyl)protected nucleosides.

TABLE 1 Photolysis Rates: 5′-Protecting Base Group Solvent Power T_(1/2)T MeNPOC dioxane 35 mW/cm²  9 sec T PYMOC MeOH ″ 10 sec T PYMOC 1:1dioxane-H₂O ″ 10 sec T PYMOC 9:1 dioxane-MeOH ″ 43 sec G^(ibu) MeNPOCdioxane 27 mW/cm² 11 sec G^(ibu) PYMOC MeOH ″ 13 sec C^(ibu) MeNPOCdioxane ″ 12 sec C^(ibu) PYMOC MeOH ″ 27 sec A^(ibu) MeNPOC dioxane ″ 12sec A^(ibu) PYMOC MeOH ″ 12 sec A^(ibu) ANMOC MeOH ″ 17 sec

Table 2 compares the coupling cycle efficiency (six cycles) of PYMOCprotected and MeNPOC protected nucleosides to a hydroxyalkylsilanatedglass support using surface fluorescence analysis.

TABLE 2 Stepwise Coupling Cycle Efficiencies: 1. Surface fluorescenceanalysis (“staircase” assay): 5′-Protecting Yield (6 steps) Base GroupNet Avg. Stepwise T MeNPOC 15 73 T PYMOC 56 91 dG^(ibu) MeNPOC 29 81dG^(ibu) PYMOC 61 92 dC^(ibu) MeNPOC 37 85 dC^(ibu) PYMOC 68 94 dA^(pac)MeNPOC 40 86 dA^(ibu) PYMOC 73 95 dA^(ibu) ANMOC 68 94

Table 3 compares the coupling cycle efficiency (six cycles) of PYMOCprotected and MeNPOC protected nucleosides to a solid support using HPLCanalysis.

TABLE 3 2. HPLC analysis (DOP#AF001; 3″ ethenodeoxyadenosine tag):5′-Protecting Yield (3 steps) Base Group Net Avg. Stepwise T PYMOC 9297.2 T ″ 93 97.6 avg 97.4 T MeNPOC 45 77 T ″ 43 75 T ″ 48 78 T ″ 40 74 T″ 48 78 avg 76.4

Front Side Photolysis Using PYMOC Protecting Groups

Two experiments were conducted using HO-PEG modified substrates on which5′-PYMOC thymidine amidite had been covalently coupled in a previoussynthesis step. Striped regions of the surface were exposed at 365 nm at35 mW/cm² for 200 seconds, under each of the following conditions: (1)dry or uncoated; (2) coated; and (3) wet (in a flow cell withWater/MeOH).

For the coated exposures, two coatings were tested: (1) 1% Triton X-100in H₂O; and (2) 0.2% Triton X-100 in 50% glycerol/H₂O. The coating wasrinsed off the substrate with dry acetonitrile in a flowcell, after theexposure. The substrates were then stained with Fluoreprime™ amidite(Pharmacia), and scanned on a confocal laser scanner. The relativeextent of photolysis for each exposure was determined from thefluorescent intensities of each stripe. The results shown in FIG. 4 showthat both coated films greatly enhanced photolysis over the dryexposure. FIG. 4, panel A shows the fluorescent scans of uncoated or dryphotolysis (left stripe), coated photolysis using 1% Triton X-100 in H₂O(center stripe) and wet photolysis conducted in a flow cell withMethanol/H₂O (right stripe). Panel B shows uncoated or dry photolysis(left stripe), coated photolysis using 0.2% Triton X-100 in 50%glycerol/H₂O (center stripe), and wet photolysis as described above(right stripe).

The Triton-water coating showed the best performance at 95% of theintensity of the wet exposure (normalized at 100%), whereas theTriton/glycerol/water coating yielded 71% of the fluorescent intensity.The two dry exposures yielded 23% and 35% of the wet exposure intensity.

Hybridization Characteristics of DNA Probe Arrays Made with 5′-PYMOCPhosphoramidites

A test array comprised of 256 decanucleotides, defined by the sequence5′-TNCNGTNCAN-3′(SEQ ID NO:1), where N=A, C, G or T, was synthesized onan Affymetrix Array Synthesizer using 5′-PYMOC-dAiBu, dGiBu, dCiBu & Tphosphoramidites. The coupling and masking procedures used to preparethe array were the same as those described elsewhere (Pease, A. C., etal. (1994) Proc. Natl. Acad. Sci. USA 91, 5022-5026), except that thephotolysis step in each cycle was carried with methanol in contact withthe surface of the substrate. For comparison, the same array was alsosynthesized by the previously described process using5′-O-(a-methyl-6-nitropiperonyloxycarbonyl-“MeNPOC”)-nucleoside monomers(photolysis in dioxane). The array was made on a glass slide which hadbeen silanated with N,N-bis(hydroxyethyl) aminopropyltriethoxysilane asdescribed previously (Pease, A. C., et al., (1994)), and then adding tothe surface a photolysable linker,MeNPOC-hexaethyleneglycol-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite. Since oligonucleotide synthesis is more efficient withthe PYMOC-building blocks, the density of “active” synthesis sites onthe substrate was reduced prior to synthesis in order to make a bettercomparison with the less efficient array synthesis of the MeNPOCbuilding blocks. This was achieved by photo-deprotecting 90-95% of thesurface MeNPOC groups by partial photolysis (i.e., exposure to light forfour half-lives), and then capping the free hydroxyl groups with amixture of tetrazole and diethoxy-N,N-diisopropylaminophosphine. Afterdeprotecting the array in 50% ethanolic ethylenediamine for 6 hours,hybridization of a complementary fluoresein-labelled oligonucleotide“target” (5′-fluorescein-ACTGGACTGAACGGTAATGCAC-3′(SEQ ID NO:2)) wascarried out at 5 nM concentration in 5×-SSPE buffer (pH 7.4), in aflowcell fixed to the stage of a scanning fluorescence microscope.Hybridization to the array was determined by scanning the surface of thesubstrate to acquire a surface fluorescence image. The hybridizationimages that were obtained are shown in FIGS. 5 a and 5 b. The congruenceof these images, demonstrates that the array fabricated with the PYMOCmonomers displayed essentially the same hybridization pattern andrelative intensities as the control array made with the standard5′-MeNPOC monomers.

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

1. A method of protecting and deprotecting a reactive group in a compound, comprising: coupling the reactive group with a second compound of the formula Ar—C(R₁)(R₂)—O—C(O)—X, wherein: Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted species of the foregoing; R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing; and X is a leaving group; provided that when Ar is 1-pyrenyl and R₁ and R₂ are H, the reactive group being protected is not an amine, to provide a protected compound with its reactive group protected by linkage to Ar—C(R₁)(R₂)—O—C(O)—; and irradiating the protected compound to provide a deprotected compound.
 2. The method of claim 1, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is hydrogen and R₂ is independently hydrogen, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.
 3. A method of protecting and deprotecting a reactive group in a compound, comprising: coupling the reactive group with a second compound of the formula Ar—C(R₁)(R₂)—X, wherein: Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted species of the foregoing; R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing; and X is a leaving group, to provide a protected compound with its reactive group protected by linkage to Ar—C(R₁)(R₂)—O—C(O)—; and irradiating the protected compound to provide a deprotected compound.
 4. The method of claim 3, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is hydrogen and R₂ is independently hydrogen, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.
 5. The method of claim 4, wherein Ar is 1-pyrenyl and R₁ and R₂ are hydrogen.
 6. The method of claim 4, wherein Ar is 9-anthracenyl, R₁ is hydrogen and R₂ is hydrogen.
 7. The method of claim 3, wherein the compound is a ribonucleoside, deoxyribonucleoside or 2′-O-methylribonucleoside.
 8. The method of claim 7, wherein the deoxyribonucleoside is linked to the Ar—C(R₁)(R₂)—O—C(O)— group via a 3′ or 5′-OH.
 9. The method of claim 3, wherein the Ar—C(R₁)(R₂)—O—C(O)— group is removed by irradiation with light.
 10. The method of claim 9, wherein the wavelength is greater than 340 nm.
 11. The method of claim 10, wherein the wavelength is about 365 nm. 